PROJECT SUMMARY This proposal aims to develop DNA cytosine deaminase enzymes as a non-destructive alternative to bisulfite for base-resolution mapping of cytosine modifications. Tailoring of the genome permits adaptation and specialization of various cell lineages derived from the same coding sequence. These epigenetic changes include modification of cytosine bases at the 5-postion of the nucleobase. The most common modification is 5- methylcytosine (mC), followed closely by 5-hydroxymethylcytosine (hmC), a product of TET enzyme-mediated oxidation of mC. Transformations involved in development, differentiation, pluripotency and even oncogenesis involve changes in the genomic patterns of mC and hmC, making it important to have robust methods to localize these modifications. The core methods most commonly used to detect these modifications involve treatment of genomic DNA with bisulfite, as the different cytosine modification states have a different propensity for bisulfite-induced deamination which can be analyzed by sequencing. Chemical deamination, however, takes place under extreme temperature and pH conditions that can degrade the vast majority of starting DNA. Therefore, the landscapes of cytosine modifications in many small or transient cell populations, like those of primordial germ cells or oocytes, have only been characterized to a limited extent. In this proposal, we will develop APOBEC-Coupled Epigenetic Sequencing (ACE-Seq), an enzymatic and non- destructive alternative for base-resolution localization of genomic hmC. This method relies on APOBEC3A (A3A), a cytosine deaminase enzyme from the AID/APOBEC family that canonically functions in innate immunity. In our preliminary work, we have demonstrated that A3A can efficiently discriminate between different cytosine modification states, offering a means for localizing modifications under mild, bisulfite-free conditions. We propose to manipulate A3A and other enzymatic components of ACE-Seq to increase its efficiency and broaden the window for discrimination between cytosine modification states. After protocol optimization, we will apply ACE-Seq in parallel with established methods to profile the hmC landscape of embryonic stem cells. Our effort will highlight the ability for ACE-Seq to function on trace quantities of DNA and to localize modifications obscured by current methodologies, offering the field a novel approach to access the epigenome of previously inaccessible genomes.